Cholera toxin, the secretory product of Vibrio cholerae responsible in part for the devastating diarrheal syndrome characteristic of cholera, activates adenylyl cyclase by catalyzing the ADP-ribosylation of Gs-alpha, the stimulatory guanine nucleotide-binding protein of the cyclase system. The toxin-catalyzed reaction is stimulated, in the presence of GTP, by approximately 20 kDa guanine nucleotide-binding proteins, termed ADP-ribosylation factors or ARFs. Rabbit polyclonal antibodies against bovine sARF II reacted with soluble and membrane ARFs but did not react with several other guanine nucleotide-binding proteins. The anti-ARF antibodies reacted with approximately 20 kDa ARF-like proteins in a variety of tissues from several species. The highest levels of immunoreactivity were observed in brain and other neural tissues. In other tissues, an immunoreactive band corresponding to SARF I was present. During rat brain development, when quantified by both immunoreactivity and function, SARF II was lowest at birth, increased somewhat by 10 days, and was maximal at 27-60 days; SARF I was unchanged. The increase of SARF II protein during postnatal development was paralleled by increased ARF 3 mRNA but not by mRNAs for five other ARFs. In the presence of GTP-gamma-S, ARF and toxin formed either an ARF-CTA complex in SDS (SDS) or self-associated ARF in DMPC/cholate, which were separated from monomeric ARF and CTA by gel filtration. Substrate specificities of ARF/toxin complexes were different from those of the monomeric proteins. The ARF 3 gene contains five exons and four introns and spans 18.3 kb. Two ARF 3 mRNAs are synthesized using alternative polyadenylation signals in exon 5. The gene appears to have multiple transcription start sites, no TATA box, no CAAT box and high G/C content in 5' promotor region. The ARF 1 gene is identical to the ARF 3 except that it has only one polyadenylation signal. The organization of the ARF 2 gene is identical to those of ARFs 1 and 3, although the promoter region is somewhat different and thus regulation of ARF 2 gene transcription may differ.